SE500219C2 - Human granulocyte proteins - Google Patents

Abstract

The invention concerns the two pure human granulocyte L1 proteins of pI 6.3 and pI 6.5, and mixtures thereof, methods for their isolation and purification, their use as marker proteins and antigenics, antisera produced against these proteins, methods for producing said antisera, the use of said antisera for the qualitative and quantitative determinatoin of L1 proteins, and test kits comprising said antisera.

Description

(20 according to SDS-polyacrylamide gel electrophoresis and gel filtration), which was very unstable but which can be used for immunization of rabbits (1). The isolation and purification methods used in reference (1) gave only very low yields of the 51,000 Dalton protein and often the protein was lost during the processes. Immunization of rabbits by injection of the 51,000 Dalton protein gave an antiserum to the L1 proteins but often also antibodies to other proteins. These antisera must be freed from undesirable antibodies by adsorption with normal human serum or normal human tissue extracts. In publication (1), unfractionated leukocytes were extracted by freezing / thawing and mechanical homogenization of the leukocytes without the addition of any enzyme inhibitors.

Publication (3) describes only analytical methods for the determination of R: 7 and R: 7 proteins; however, no insulation or purification procedures are described.

An object of the present invention is to provide gentle and efficient methods for extracting and purifying the L1 proteins from leukocytes which enable their characterization and production of monospecific antisera or antibodies to these proteins. A further object of the present invention is to provide test systems comprising such antisera and / or such purified L1 proteins, optionally labeled with a radioactive isotope or with an enzyme suitable for quantitative testing of L1 proteins in body fluid. cows or cell or tissue extracts.

It has surprisingly been found that the L1 proteins can be obtained in a pure and stable state if only granulocytes are used as the source and if the granulocytes are destroyed under such conditions that the lysosomes inside the granulocytes are not destroyed simultaneously. This can be achieved by pressure homogenization of granulocytes. the cells in the presence of an enzyme inhibitor and an agent that stabilizes the lysosomal membranes.

Accordingly, the present invention relates to pure p1,6,3 and p1,5-L1 proteins and mixtures thereof, methods for extracting them from granulocytes by pressure homogenization of the granulocytes in the presence of an enzyme inhibitor and an agent which stabilizes the lysosomal membranes and their purification by various methods, the use of these proteins for the production of monospecific antisera or antibodies, the procedure for the production of such monospecific antisera or antibodies by immunization of experimental animals with the L1 proteins, monospecific antisera and antibodies per se, the use of such antisera or antibodies and / or the purified L1 proteins for qualitative and / or quantitative determination of the L1 proteins in cells, tissues and body fluids and test systems in - including these pure L1 proteins, antisera and / or antibodies, optionally labeled with radioactive isotopes or with enzymes.

The invention relates in particular to pure p1,6,3 and p1,5-L1 proteins, mixtures thereof and salts thereof. Salts of the L1 proteins are especially non-toxic pharmaceutically acceptable salts or otherwise those which can be used for the precipitation or purification process of the L1 proteins. Such salts are, for example, metal salts, such as alkali metal or alkaline earth metal salts, for example sodium, potassium, magnesium or calcium salts, or zinc salts and the like, or ammonium salts with ammonia or with primary, secondary or tertiary amines, for example salts with aliphatic, cycloaliphatic, cycloaliphatic-aliphatic or araliphatic primary, secondary or tertiary amines, such as with lower mono-, di- or tri-lower alkylamines, e.g. triethylamine, hydroxy-lower alkylamines, for example 2-hydroxyethylamine, bis- (2-hydroxyethyl) amine or tris- (2-hydroxyethyl) amine or with basic aliphatic esters of carboxylic acids, for example ethylenediaminetetraacetic acid-1α-alkyl esters, for example tetraethyl ester and the like.

Additional salts are acid addition salts with inorganic acids, especially mineral acids, such as hydrochloric acid, sulfuric acid, phosphoric acid or with organic acids, such as carboxylic, sulfonic or sulphoic acids, optionally substituted lower alkanecarboxylic acids, for example acetic acid, propionic acid, glycolic acid, maleic acid, maleic acid malonic acid, fumaric acid, tartaric acid, citric acid or optionally substituted aromatic acids, I for example benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-amino-salicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid, isoniconicoric acid, or amino amino acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalenesulfonic acid or ascorbic acid and the like.

Both proteins are characterized by their isoelectric points at pH 6.3 and pH 6.5, respectively. The molecular weight of both proteins determined by gel filtration and density gradient ultracentrifugation is the same and is about 36,500 Daltons. By SDS electrophoresis on polyacrylamide gel, the proteins are shown to consist of three polypeptides with approximately identical molecular weights of 12,500 Daltons. Each of these polypeptide chains exhibits the following amino acid sequence beginning at the N-terminal end: Meth-Leu-Thre-Glu which are detected by the standard Edman method.

The electrical addition and availability of antigenic sites on the L1 proteins are greatly affected by the absence or presence of calcium ions. The proteins react much more strongly as antigens in vitro in the presence of calcium ions.

The thermal stability of the L1 proteins is high at neutral and alkaline pH. Boiling for 15 minutes in aqueous 5 mM EDTA, pH 8.5, preserves 90% of the protein. The L1 protein is partially precipitated with ethanol at acidic pH, for example between about 4 and 5.

The L1 proteins show a characteristic distribution in the human body, being found in large amounts in neutrophilic granulocytes, monocytes and squamous epithelial cells in addition to normal skin. They are also found in many squamous epithelial-type cancer cells and in certain leukemia and lymphoma cells, especially those of the myelogenous leukemia and monocytic lymphoma type.

The L1 proteins are found in a predominant number in squamous cell carcinoma of the urinary tract and lungs. The L1 proteins are also found in normal body fluids in relatively low concentrations in healthy individuals n increasing concentrations in conditions with increasing degree of inflammation. In white blood cells the L1 proteins are present until 90% of the L1 proteins can be used as marker proteins, especially in radiolabeled form, for example with 1125 or P32, for qualitative and / or quantitative determination of the L1 protein content in body fluids, cells and tissues. .

The L1 proteins are good immunogens in animals, for example in the cytosol fraction. rabbits.

The invention further relates to a process for the preparation of the pure pI 6.3 and pI 6.5 L1 proteins, mixtures thereof and salts thereof, characterized in that human granulocytes are decomposed in a suitable buffer system, insoluble cell material is removed and the supernatant is removed. a) isoelectric focusing and elution of the bands containing pI 6, 3- and / or p1 6,5-L1 proteins with a buffer, or b) anion exchange chromatography with an EDTA-containing buffer at a pH of about 8.5 and eluting the desired LI proteins with a buffer containing calcium ions, or c) affinity chromatography, or d) preparative agarose gel electrophoresis, or e) cation exchange chromatography, or f) gel filtration, or g ) reversible precipitation with Zn ++ any combination of steps a) to g), liberation of the eluate from any ampholytes and / or salts, concentration of the resulting solution of pure L1 proteins if desired, lyophilize of the obtained concentrated solution and, if desired, transformation of the obtained L1 proteins into a salt or transformation of the obtained salt into the free or the proteins.

The granulocytes used as a source for the extraction of L1 proteins are obtained from fresh human blood by any conventional method, for example by dextran sedimentation and density gradient centrifugation.

The buffer system in which the granulocytes burst contains an agent which prevents simultaneous bursting of the lysosomes, such as glycerol, dextrans and preferably sucrose, in a concentration sufficient to stabilize the osmotic pressure before and after bursting of the cell walls. For example, sucrose was used in a concentration of between 0.27 and 0.34 M.

The buffer system further contains enzyme inhibitors which prevent the digestion of the L1 proteins, such as diisopropyl fluorophosphate (DFP), soybean trypsin inhibitor, Trasilol, mercury benzoate, pepstatin A and preferably phenylmethylsulfonyfluoride (PMFS) and ethylene dihydric or ethyl acetic acid or dihydric acid.

The blasting of the cell wall can be carried out in a conventional manner, for example mechanically, e.g. with a rotary mixer, a mortar (Potter-Elvelyem Method), by means of ultrasound, freezing and thawing or combinations of these methods. According to a preferred method, the granulocytes burst by pressure homogenization.

After disruption of the granulocytes, insoluble materials such as non-disrupted cells, cell debris and lysosomes are removed, for example by ultracentrifugation. The L1 proteins are obtained in pure form from the crude granulocyte extract by any of the following purification procedures. a) Isoelectric focusing: The isoelectric focusing is preferably carried out in a granular gel, such as beads of agarose or polymerized dextran containing a suitable mixture of commercially available ampholytes to provide a stable pH gradient of from about pH 5 to 8 during the process. The isoelectric focusing is performed with a maximum voltage of about 25 V / cm for about 10-20 hours at a temperature of about S to 150. A preferred gel is Ultrodex® and the preferred ampholyte is Ampholine®.

The sample is preferably applied to the cathode side of the gel.

The localization of the L1 protein bands can be carried out by pH measurements along the gel surface or by staining filter paper prints, of the top surface, for example with "coomassie" blue.

The bands containing the L1 proteins are scraped off and eluted by a suitable buffer, containing EDTA at a pH of between about 7.5 and about 9, preferably at a pH of about 8.5.

The eluate is freed from the ampholytes and salts by dialysis and / or gel filtration and concentrated by ultrafiltration and / or lyophilization. b) Anion exchange chromatography: used according to this procedure contains, for example, tertiary or quaternary amino groups and consists, for example, of diethylaminoethylcellulose, e.g. diethylaminoethyl-Sepharose or QAE-cellulose (quaternary aminoethylcellulose). The granulocyte extract containing the L1 proteins is applied to the column in a low ionic strength buffer containing EDTA and having a pH of about 8.5.

The L1 proteins are specifically eluted by means of a calcium ion-containing buffer, for example a buffer containing 30 mM barbital Anion exchange materials as an and 5 mM calcium chloride. c) Affinity chromatography: A third method for purifying the L1 protein is based on affinity chromatography. This can be done on regulated porous glass beads having a large surface area, for example about 50 m2 / g. The glass beads are placed in a column and the crude granulocyte extract is applied to a suitable low molarity buffer containing calcium ions at neutral or slightly acidic pH, for example about pH 6-7. The L1 proteins are eluted with an alkaline buffer of about pH 7.5-8.5 containing about 50 mM 10 15 20 ZS 30 35 40 EDTA and polyethylene glycol or lithium bromide.

According to another affinity chromatography method, an anti-L] antibody column was used. The L1 proteins are adsorbed to the antibodies while the other proteins are washed out with a buffer. Thereafter, the L1 proteins are eluted with a saline solution, such as an aqueous buffer of about pH 3.5, containing a salt, such as sodium chloride or sodium iodide. d) Preparative agarose gel electrophoresis: Flatbed agarose gels of suitable dimensions were used containing 75 mM barbital / 2.5 mM EDTA, buffer, pH 8.5. Electrophoresis is performed at 4 V / cm for 10-18 hours. The L1 protein bands are located by staining the filter paper imprint of the gelovan surface. The L1 protein bands are found in the xz-globulin region and are eluted by freezing / thawing and centrifuging the relevant strips of the agarose gel. e) Cation exchange chromatography: performed on cation exchange resins, such as sulfopropyl-substituted cellulose or polymerized dextran. The crude granulocyte extract is applied to the column after mixing with a low ionic strength buffer containing calcium ions and having a pH of about 6.

Non-adsorbed proteins are washed away with the starting buffer and the L1 proteins are eluted with a buffer containing EDTA and cation exchange chromatography increasing concentrations of sodium ions. f) Gel filtration: For the gel filtration, Sephadefß G75 to G200 agarose gels (Sepharose gels), such as Ultra gel or polyacrylamide gel beads, such as Bio-Gel P-60, were used.

The column is preferably a long one. The column length should be about 50 times the diameter. The crude granulocyte extract is applied as such and the L1 proteins are chromatographed. For packing the column and processing, a buffer containing preferably 0.1 M sodium citrate, 2.5 mM EDTA-K2 and 0.25 mM thimerosal, pH 8.5 was used. The L1 proteins are found in a fraction corresponding to a molecular weight of 35,000 to 40,000 Daltons. g) Reversible precipitation with the Zn ++ Li proteins is quantitatively precipitated from aqueous solutions with neutral or alkaline pH by treatment with Zn ++ ions, especially in the form of water-soluble zinc salts, such as zinc acetate or zinc chloride.

For example, an aqueous solution containing the L1 proteins can be treated at pH about 8.5 with an aqueous solution of about 10 mM to 100 mM zinc acetate. The precipitated L1-zinc salt is recovered, washed if necessary with the aqueous buffer solution, treated with a complexing acid, for example an approximately 100 mM aqueous dipotassium ethylenediaminetetraacetic acid solution of pH 8.5 and to recover the the free L1 proteins, the solution is filtered through an ion exchange column, for example Pharmacia PD-10, and lyophilized.

The salts of the L1 protein are formed by conventional methods, for example by treatment with the relevant ions, for example a base, an acid or a salt containing the desired ions or by ion exchange treatment. A highly insoluble manganese salt can be prepared, for example, by adding Mn ++ ions, for example in the form of an approximately 10-mM to 100-mM aqueous solution of manganese acetate or some other water-soluble manganese salt, to an aqueous solution of L1- the proteins.

The invention also relates to monospecific antisera to the L1 protein and the antibody containing the immunoglobulins, especially those formed by rabbits.

Monospecific antisera are prepared by conventional methods, for example by weekly injection of the pure L1 proteins with complete Freund's adjuvant, for example subcutaneously or intramuscularly, at doses of about 100 pg per rabbit per week for about 8-16 weeks. Serum is then collected from the animals, for example by venous section, and clotted. Instead of rabbits, other non-human warm-blooded animals, especially mammals, can also be used, e.g. sheep, goats, rats, mice, horses and the like.

The monospecific antibody-containing globulin fraction can be isolated from serum by conventional methods, for example by ion exchange chromatography.

For the preparation of these monospecific anti-L1 antibodies, antiserum containing the monospecific antibody or globulin fraction thereof is driven through a column where the purified L1 proteins have been conjugated to a suitable material, such as agarose or cellulose gel. After washing off non-adsorbed proteins, the anti-L1 antibody molecules are eluted with a buffer having a pH of about 3.5 containing sodium chloride or sodium iodide.

The relevant fractions are adjusted to a pH of about 7, dialyzed and concentrated.

The anti-L1 antibodies, either purified or in crude form, such as in the form of antiserum or globulin fractions thereof, were used for the qualitative or quantitative methods for detecting L1 proteins. in cells, tissues or body fluids.

Such methods include gel precipitation, nephelometry, radioimmunoassay, enzyme immunoassay, immunofluorescence, and immunoperoperidase cytohistochemical methods in a conventional manner.

The invention further relates to test systems comprising those monospecific anti-L1 antibody or anti-L1 antibody-globulin fraction or anti-L1 antibody-containing antiserum.

Such test systems are of the conventional type and are intended for simple radioimmuno-diffusion, latex agglutination, spot test-radioimmunoassay, enzyme-immuno-assay, immunofluorescence test or enzyme-immunohistochemical test.

Such systems may contain, in addition to pure or crude anti-L1 antibodies, dried or solubilized in a suitable buffer, pre-prepared gels, latex, polystyrene, formalin-stabilized animal red blood cells or similar particles, enzymes such as radish peroxidase, alkaline phosphatase , enzyme substrates, fluorescence or enzyme-conjugated antibodies and materials suitable for the separation of antibody-bound L1 molecules from free fl molecules, such as killed strains of Staphylococcus aureus belonging to the strains forming Staphylococcus protein A, charcoal free as the protein but not the antibody-protein complex) or a second antibody such as goat or anti-rabbit immunoglobulin or L1 proteins labeled with radioactive isotopes such as 1125 or with enzymes such as radix peroxidase or alkaline phosphatase. The system may also contain carriers to which monospecific anti-L1 antibodies are attached to a surface, for example the inner surface of test tubes, the surface of glass or plastic beads, pieces of filter paper, cellulose acetate membranes or similar materials, and buffers for such methods.

In the above and below, the following abbreviations are used: PMSF phenylmethylsulfonyl fluoride PAGE: polyacrylamide1 electrophoresis SDS: sodium dodecyl sulfate IEF isoelectric focusing SRID single radioimmunodiffusion NHS normal human serum ACD acid citrate dextrose 10 15 21 25 25 Dophylate 40 15 20 25 30 35 35 40 EDTA ethylenediaminetetraacetic acid EDTA-K2: dipotassium salt of EDTA DEAE diethylaminoethyl RIA radioimmunoassay PBS phosphate buffered saline PASA protein A-containing Staph. aureus Example 1: Isolation of granulocytes a) A mixture consisting of 435 ml of fresh human blood, 65 ml of an aqueous solution containing 2.2% trisodium citrate dihydrate, 0.8% anhydrous citric acid and 2.45% dextrose, 250 ml high molecular weight dextran (Dextraven® 150) and 23 ml of a solution containing 2.5 mM citric acid, 45 mM trisodium citrate, 180 mM glucose and 15 mM sodium chloride are allowed to settle for 1 hour at 4 ° C. The supernatant layer is collected and centrifuged at 200 x g for 20 minutes at 4 ° C. The sediment is treated with 50 ml of ice-cold distilled water, whereby the red blood cells are lysed. After 30 seconds, isotonicity is restored by the addition of 16.5 ml of ice-cooled 0.6 M sodium chloride. Remaining leukocytes are washed twice with isotonic saline. The granulocytes are separated from the mononuclear cells by centrifugation on Lymphopre fl g) [density gradient centrifugation material consisting of 9.6% (w / v) sodium metrizoate solution containing 5.6% (w / v) of an erythrocyte aggregating polysaccharide ] followed by centrifugation at 800 xg for 20 minutes at ZOOC. This method gives a yield of about 5 x 108 cells, of which about 95% are granulocytes. b) Granulocyte extraction The cells obtained as described in a) are resuspended in 0.27 M sucrose containing 10 mM PMSF, 2.5 mM EDTA-K 2 and 0.25 mM thimerosal with the addition of Tris buffer to pH 8.5. Thereafter, the cells of French and Holm (4) are homogenized by exposure to 28 kp / cm 2 compressed air for two periods of 2 minutes while allowing them to be expelled from the pressure chamber at a rate of one drop per second. During this procedure, the cell containers are kept in wet ice. About 90% exploded and after centrifugation at 100,000 x g for 1 hour, about 90% of the total L1 protein was found in the clear supernatant fluid. The latter is called the crude granulocyte extract. of the cells The total protein content is about 3 mg / ml, of which 30% is Lqnotenær. 10 15 20 25 30 35 40 “» 500 219 c) Preparative isoelectric focusing (IEF) For preparative IEF, a gel plate measuring 110 mm x 240 mm x 5 mm consisting of Ultrade (Dextran G75, refined) containing 2% (w / w) was used. volume) Ampho1inÅÉ) pH 5-8 (a mixture of synthetic ampholytes with buffering capacities at the isoelectric points therefore). The samples [B ml of the supernatant according to b)] were mixed with an equal volume of Ultrade gel and applied 5 cm from the cathode to the gel plates. Separation is performed for 18 hours with a maximum of 25 V cm ', 15 mA and 10 W.

The protein bands were located both by "coomassie" blue staining of filter paper 8VIïyCk of the gel plate and gaum1pH measurements of the gel surface. The L1 proteins in the range of pH 6.3 and 6.5 were eluted with an equal volume of an aqueous solution containing 0.1 M sodium acetate, 2.5 mM EDTA-K 2 and 0.25 mM timerol, pH 8.5, by centrifugation at 2000 xg for 20 minutes through a 0.45 Pm Millipore filter. The ampholytes were removed by dialysis against the elution buffer overnight. The remaining protein- and buffer-containing solutions were concentrated by ultrafiltration on Minocon cells (semipermeable membrane). The yield of these processes is about 20% of the L1 protein applied to the gel for preparative IBF. 2.5 ml of this solution was applied to a Pharmacia PD-10 column (Sephade G25 M) equilibrated with distilled water and the L1 proteins were eluted with 3.5 ml of distilled water and lyophilized to give 1 mg of pure L1 protein p6 , 3 and 6.5.

The L1 protein with p1 6.3 and p1 6.5 can also be purified separately by this procedure to give about 0.67 mg of the L1 protein with p1 6.5 and about 0.33 mg of the L1 protein p1 6.3 .

Example 2: ~ a) The process takes advantage of the dramatic change in the isoelectric point of the L1 protein when it is allowed to take up calcium ions (2). The protein in crude granulocyte extracts binds to an anion exchanger, for example the diethylaminoethyl type, at alkaline pH and with a buffer containing ethylenediaminetetraacetic acid in sufficient amounts to bind calcium ions.

The L1 proteins can then be eluted specifically by adding calcium chloride to the buffer to ensure a final concentration of about 5-10 mM per liter.

Crude granulocyte extracts are prepared as described in Example 1. Their total protein content is about 2-5 g per liter, of which about 60 percent is L1 protein.

DEAE-Sephacel (from Pharmacia, Sweden) was equilibrated with 60 mM barbital buffer (7.7 g diethyl barbituric acid and 10.5 g of its sodium salt in 1 liter of water) containing 1 g / l EDTA-K 2, pH 8.5 and packaged on a chromatography column with the dimension 1.5 cm x 5 cm, ie a volume of about 9 ml.

The sample, about 10 ml of crude granulocyte extract diluted with 30 ml of EDTA-K2 barbital buffer, pH 8.5, was applied with a peristaltic pump, giving a flow rate of about 2 ml / min.

Non-adsorbed proteins were washed out with 100 ml of barbital buffer. Before eluting the L1 proteins, the column was also washed with 30 ml of barbital buffer without EDTA-K2. The L1 proteins were then eluted with 60 mM barbital buffer containing 5 mM CaCl 2, pH 8.5.

Pig. Figure 1 shows the overall protein and L1 protein elution profile for such ion exchange chromatography and Figure 2 shows the protein band patterns when crude leukocyte extract and purified L1 protein are subjected to analytical agarose gel electrophoresis. It can be seen that the purified L1 protein yields two larger bands as previously described corresponding to pI 6.3 and pI 6.5. The amount of L1 protein eluted with calcium-containing buffer is close to 50% of that applied to the sample and found in a total volume of about 20 ml. This solution can be conveniently concentrated by ultrafiltration using Millipore CX-10 membranes. This leads to some protein loss depending on whether EDTA is added or not to prevent L1 protein from binding to the membranes. Even without the addition of EDTA, a total yield of about 35% can be achieved.

As shown in Figure 1, some L1 protein is eluted with 60 mM barbital buffer containing 5 mM CaCl 2 and 125 mM NaCl, pH 8.5, but this protein yields bands that are predominantly anodic and cathodic of the predominant L1 bands. and may represent partially I denatured or complexed L1 protein in addition to various proteins.

Large scale methods The method described above can be successfully augmented for handling 50 ml of raw granulocyte extract. This is achieved by using a column containing about S0 ml DEAE-Sephace (about 2.5 cm x 10 cm) and proportional increases in the volume of the same buffer substances as indicated above. b) The fact that a reversible precipitation of the L1 proteins can be achieved by the addition of 10 mM Zn ++ can be used. To 10 ml of crude leukocyte extract are added 90 ml of barbital buffer, 75 mM, pH 8.5, and 10 ml of a 100 mM Zn acetate solution in water. The mixture is washed several times, for example five times, with 75 mM barbital buffer. pH 8.5, with 10 mM Zn acetate. Finally, the precipitate is washed with 75 mM barbital buffer, pH 8.5, and dissolved by dropwise addition of a solution of 100 mM EDTA-K2 in water, the pH is adjusted to 8.5 by 5 M sodium hydroxide. This protein solution is applied to a Pharmacia PD-10 column equilibrated with distilled water and the L1 proteins are eluted with 3.5 ml of distilled water and lyophilized to give about 10-20 mg of pure 210 z L1 proteins.

Example 3. Preparation of antiserum to purified L1 A solution containing about 1 mg / ml of L1 protein of pI 6.3 and pI 6.5 (obtained according to Example 1 or 2) in 0.1 M sodium acetate, 2.5 mM EDTA, 0.25 mM thimerosal, pH 8.5, was homogenized with Freund's complete adjuvant. A volume of this mixture containing approximately 100 pg of L1 proteins was injected at several subcutaneous sites in rabbits once a week for 6 weeks.

The rabbits were bled [fi. Blood by venipuncture. The blood was allowed to coagulate at room temperature overnight. Serum was recovered and tested for L1 antiserum activity.

Normally, such antiserum can be used in a 1:32 dilution for a single radioimmunoprecipitate and a 1: 2000 dilution for radioimmunoassay.

Example 4: Radioimmunoassay Test System The test system contains (for 100 tests): 1 ampoule of 2 ml lyophilized anti-L1 serum obtained according to Example 3 1 bottle of 100 ml RIA buffer: 50 mM sodium chloride, 0.2% bovine serum albumin, 0.2% sodium azide, adjusted to pH 7.4; 1 rubber-filled ampoule of 5 ml aqueous solution of radioactive iodine-labeled L1 proteins adjusted to give S00 cpm / ml (according to the Bolton-Hunter labeling method); 1 bottle of 100 ml of an aqueous suspension of formalin-treated Staphylococcus aureus (PASA = protein A-containing Staph. Aureus of Cowan I strain) in phosphate buffered saline; 1 bottle of 2 ml RIA buffer solution containing 500 ng L1 protein (reference solution). 10 15 20 25 30 35 40 500 219 '4 Method for quantification of L1 proteins according to RIA A series of standards was prepared by doubling the dilution of the provided L1 reference solution down to 7.8 ng / ml with RIA buffer. In a series of test tubes, 25 μl of patient plasma diluted 1:30 in RIA buffer or standard reference solution was pipetted. To this was added 20 μl of anti-L1 serum and the mixtures were incubated at 37 ° C for 30 minutes. Then 50 μl of the solution with radiolabeled L1 protein was added, which was followed by incubation at 37 ° C for 1 hour. To each tube was then added 1 ml of suspended Staph. aureus suspension and centrifuged at 2000 x g for 10 minutes. The supernatant was sucked off and the radioactivity in the sediment was measured. The percentage of the radioactivity in relation to the initially added radioactivity was set aside for the L1 reference standard dilutions so that the reference curve could be plotted. The values from the patient samples can be read from this curve.

Example S: Test system for nephelometric determination of L1 proteins in solution The test system contains: 1 ampoule of 1.5 ml anti-L1 antibody solution in buffer of pH 7, É (sodium acetate buffer containing 5 mM calcium chloride) with a titer of about 1 : 32 tested by double immunodiffusion in agarose gel; 1 bottle of 20 ml dilution buffer as above; 1 ampoule of 0.5 ml of a solution of L1 protein at a concentration of 400 pg / ml Anti-L1 serum was diluted 1:15 with dilution buffer. Six standards of L1 protein (1: 1 to 1:64) were prepared by double dilution of the standard L1 solution. Then, 250 μl of antibody dilution and 10 μl of sample or diluted L1 standard were mixed in nephelometer cuvettes and incubated for 10 minutes at room temperature. The optical density was read on the nephelometer.

Example 6: Test system for simple radioimmuno-diffusion analysis of L1 proteins in solution The test system contains: 1 or more pre-prepared agarose gels containing 2% of homogeneously distributed anti-L1 antibody solution with a titer of about 1:32 when tested by double diffusion in gel and 0.1 M sodium acetate, 5 mM calcium chloride, 0.25 mM thimerosal, pH 8.5; 10 15 20 25 30 35 40 15 500 219 1 ampoule 0.1 ml L1 protein solution with a concentration of 300 pg / ml. 'A __4 The agarose gel was provided in a closed chamber to prevent drying of the gel. The gel showed 18 pre-prepared wells for application of 10 μl of standard solutions and the samples to be tested.

Test system for latex agglutination assay for a semi-quantitative determination of L1 protein Example 7: The test system contains: a) 1 bottle and 2 ml of a suspension of latex particles of 50 Pm diameter to which purified L1 protein has been fixed; b) 1 vial and 2 ml of an anti-L1 antibody buffer solution agglutinating with the above latex particles, having a titer of 1: 3 to 1: 4; c) 1 bottle and 0.5 ml of a buffer solution of the L1 protein at a concentration of 50 pg / ml.

A drop of a), b) and the patient sample to be tested are mixed on a black disc and observed for agglutination for 5 minutes. Lack of agglutination means an L1 concentration of over 1000 ng / ml. In the positive control solutions, a), b) and c) are mixed and no agglutination should be developed. Example 8: Spot test test system The test system contains: 10 pieces of cellulose acetate measuring 5 mm x 50 mm with 2 spots with a diameter of 4 mm of dried specific anti-L1 antibody located 10 mm and 30 mm from one end; A) 1 vial of 2 ml of a buffer solution containing specific anti-L1 antibodies conjugated to radish peroxidase; b) 1 bottle of 10 ml of an aqueous solution of Tris buffer / isotonic saline, pH 7.4, containing 1 mg / ml diaminobenzidine (DAB); c) 1 bottle of 1 ml of an aqueous buffer solution of L1 protein at a concentration of SO pg / ml For testing, apply one drop of the sample solution to the first spot and one drop of solution c) to the second spot.

After 10 minutes, the test strip was washed in Tris buffer / saline for 5 minutes. Then a drop of solution a) is applied to each stain and left for 10 minutes at room temperature and the strip is washed again for 5 minutes in Tris buffer / saline.

To 1 ml of solution b) is added 5 μl of 30% hydrogen peroxide.

A drop of this mixture is applied to each spot. A distinct brown staining occurs after 3 minutes, indicating the presence of L1 proteins in the sample.

By serial dilution of the sample, the test can be performed semi-quantitatively.

Example 9: Test system for L1 proteins by enzyme-immuno-assay The test systems contain: 1 microtiter tray with 5 x 10 wells made of plastic material, the inner surface of the bottom and the lower part of the wells being coated with specific anti- L1 antibodies; a) 1 vial of 2 ml of a buffer solution containing specific anti-L1 antibodies conjugated to radish peroxidase; b) 1 bottle with 10 ml of an aqueous solution of Tris buffer / isotonic saline, pH 7.4, containing 1 mg / ml of diaminobenzidine (DAB); c) 1 bottle fl ßd 1 ml of an aqueous buffer solution of L1-protein at a concentration of SO pg / ml.

Test procedure: SO μl of this sample solution or of solution c) is added to the wells and incubated at room temperature for 1 hour. The wells were then washed with Tris buffer / saline containing 0.5% bovine serum albumin. To each well was added 50 μl of solution a) and incubated for 1 hour at room temperature. The wells were washed as above. To 1 ml of solution b) was added 5 μl of 30% hydrogen peroxide and SO μl of this mixture was added to each well. The tray was incubated for 30 minutes at room temperature and the color intensity was determined for each well photometrically.

Explanations of the figures Pig. 1: Chromatographic profile of 9 ml crude leukocyte extract on a DEAE-Sephacel column, 1.5 cm x 5 cm. The total protein content was determined by UV light absorbance at 280 nm and the L1 concentration by a simple radioimmunodiffusion.

Fig. 2: Protein band pattern of crude granulocyte extract (A), s mm caciz-fraction-.ionefi- (B) and 1zs mn Naci-fraction (c) by agarose gel electrophoresis in barbital buffer, 75 mM, with 2.5 mM EDTA-K2, pH 8.5. Fraction B contains the two pure L1 proteins pI 6.3 and pI 6.5. 17 '500 219 Literature references: (1) (2) (3) Magne K. Fagerhol, Inge.Dale and Terje Andersson, Scand. J. Haemancu. (1980) 24, '393-398.

Dale, T. Andersson, Bull. 1980, 16 (suppl) 273 281.

M.K. Fagerhol, I.

Physiopath. resp. europ.

Karen E. Wíllard, Anne Karíne Thorsrud, Eimar Munthe and Egil Jellum, Clin. Chem., Vol. 28, No. 4, 1067-1073 (1982).

French. P.C. (1947) Thromb. Diath. 32, and Holm, R.A., Haemorrh. 432-440.

Claims (9)

10 15 20 25 30 35 40 500 219 i '.ß Patent claims 1. Pure 6, §- and pI 6,5-L1 proteins, characterized in that they have a molecular weight of 36,500 Daltons and consist of three polypeptide subunits; and mixtures and salts thereof. Protein according to claim 1, characterized in that it consists of the pI 6,3-L1 protein or salts thereof. A protein according to claim 1, characterized in that it consists of the pI 6,5-L1 protein or salts thereof. Process for the preparation of pure pI 6,3 and pI 6,5-L1 proteins, mixtures or salts thereof according to claim 1, characterized in that human granulocytes are burst into a suitable buffer system containing an agent which prevents a simultaneous bursting of lysosomes, at a concentration sufficient to stabilize osmotic pressure before and after the cell wall burst, insoluble cellular material is removed and the supernatant is subjected to a) isoelectric focusing and elution of the bands containing pI 6.3- and / or pI 6,5-L1 proteins with a buffer, or - b) anion exchange chromatography with an EDTA-containing buffer at a pH of about 8.5 and elution of the desired L1 proteins with a buffer containing calcium ions , or c) affinity chromatography, or d) preparative agarose gel electrophoresis, or e) cation exchange chromatography, or f) gel filtration, or g) reversible precipitation with Zn **, or any combination of steps a) to g) , the eluate is freed from any ampholytes and / or salts, the solution of pure L1 proteins obtained is concentrated, which L1 proteins have a molecular weight of 36500 Daltons, determined by gel filtration or density gradient ultracentrifugation; if desired, the obtained concentrated solution is lyophilized, and if desired, the obtained L1 proteins are transformed into a salt, or the obtained salt is transformed into the free proteins- Da. 15 W 500 219 ' Use of the L1 proteins according to claim 1 as marker proteins or as immunogens in animals. A Monospecific anti-L1 antisera and monospecific anti-L1 antibodies are characterized in that they are prepared with pure L1 proteins according to any one of claims 1-3. A process for the preparation of anti-L1 antisera and anti-L1 antibodies according to claim 6, characterized in that animals are treated with pure L1 proteins according to claim 1 and that antisera or antibodies are obtained by conventional methods. Use of anti-L1 antisera and anti-L1 antibodies according to claim 6 for the qualitative or quantitative determination of L1 proteins in cells, tissues or body fluids. A test system, characterized in that it comprises monospecific anti-L1 antibodies, anti-L1 antibody globulin fractions or anti-L1 antisera according to claim 6 {